The dynamic cellular control of G protein-coupled receptor (GPCR) trafficking impacts many functional aspects of the protein. Accumulating evidences have suggested the endocytosis and intracellular trafficking of GPCR not only are critical in the desensitization and resensitization of the receptor but also these processes participate in generating alternative signals. The trafficking of the opioid receptors is also under stringent cellular control, and appears to have a role in the cellular responses to the chronic opioid agonist treatment. Previous reports from our laboratory and others have indicated that mu and delta-opioid receptors, though structurally homologous, have different intracellular trafficking patterns. The agonist-induced internalized mu-opioid receptor can be resensitized and recycled back to the cell surface, while the delta-opioid receptor is directed to the lysosomal degradation pathway. Receptor chimeras studies suggest that the carboxyl tail domains of these two receptors are responsible for such trafficking patterns. However, as demonstrated in our Preliminary Data, carboxyl tail domains participate but not sufficient in directing the trafficking of the opioid receptors in a neuronal cell model, neuroblastoma Neuro2A (N2A) cells. An interaction between a dileucine motif within the 3 rd intracellular loop of the delta-opioid receptor with the carboxyl tail is needed for directing the receptors to lysosomes. Thus, we hypothesize that in addition to the linear receptor sequence, multiple three-dimensional sequences generated by covalent modifications of the receptors such as phosphorylation and ubiquitination are involved in directing the opioid receptor traffic. The scaffolding of the proteins in the endocytic pathways with the modified receptors will determine the direction of the receptor traffic. Hence, in the current studies, we propose to identify: (a) the linear receptor sequences that participate in the intracellular trafficking of the opioid receptor; (b) the role of ubiquitination and phosphorylation on generating the three-dimensional sequence in the intracellular trafficking of the receptor; and (c) the tmns-endocytic proteins that are critical in the recycling and lysosomal trafficking of the receptor. Truncation, deletion, single amino acid and random mutational analyses of the mu and delta-opioid receptor sequences will be carried out to delineate the primary sequences involved. Yeast two-hybrid screens and proteomic approaches using mass spectrometry will be used to identify the trans-endocytic proteins involved. Dominant negative mutants of these proteins will be used to demonstrate their roles in receptor trafficking. By establishing the itineraries of the/,- and 0-opioid receptors, and by identifying the endocytic protein complexes in various pathways in N2A cells, we will be able to illuminate the dynamic nature of the cellular control and elucidate the significance of the receptor trafficking in the opioid agonist function in neurons.